We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting to these compartments is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats. Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (GGAs), and the heteropentameric retromer complex. Proper sorting also requires the function of other components of the trafficking machinery that mediate vesicle tethering and fusion, such as the heterotetrameric GARP complex. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects (Hermansky-Pudlak syndrome) or pathogens'(HIV-1) exploitation of these proteins. The AP-4 complex is the most-recently discovered and least well-understood of the family of AP complexes. In recent work, we have found that AP-4 with the cytosolic tail of the Alzheimers Disease (AD) amyloid precursor protein (APP). Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network (TGN) and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These findings establish AP-4 as a novel regulator of APP processing and trafficking, and as a potential risk factor for AD. The retromer complex is a sorting device that mediates retrograde transport from endosomes to the TGN. This function is essential for many important physiological processes in higher eukaryotes, including lysosomal enzyme sorting, processing of APP, and formation of morphogen gradients during development. The retromer comprises a membrane-binding subcomplex made up of two sorting nexin (SNX) subunits and a cargo-recognition subcomplex composed of Vps26, Vps29 and Vps35. In previous studies, we discovered a role for the retromer complex in the retrieval of mannose 6-phosphate receptors from endosomes to the TGN and showed that this retrieval is essential for acid hydrolase sorting to lysosomes. In collaboration with James Hurley (NIDDK), we also solved the crystal structure of the Vps26-Vps29-Vps35 complex. Over the past year, we found that the recruitment of the Vps26-Vps29-Vps35 subcomplex to membranes involves, in addition to the SNX subcomplex, the small GTP-binding protein Rab7. Perturbation of either the SNX subcomplex or Rab7 results in dissociation of the Vps26-Vps29-Vps35 subcomplex from membranes. In turn, this leads to missorting of acid hydrolases and consequent accumulation of undegraded materials in lysosomes, a phenotype characteristic of lysosomal storage disorders. Endosomal transport carriers formed by the action of retromer must dock at and fuse with the TGN in order to deliver their cargo. We previously showed that GARP, a multi-protein complex originally described in yeast, plays such a role in mammalian cells. Interference with GARP blocks the delivery of cargos such as mannose 6-phosphate receptors and Shiga toxin from endosomes to the TGN, indicating that GARP has a general role in retrograde transport. A mutation in one of the GARP subunits, Vps54, was recently identified in the Wobbler mouse mutant, an animal model of amyotrophic lateral sclerosis. We have found that the Wobbler mutation does not prevent the function of GARP in retrograde transport, suggesting that the disease is likely due to a subtle defect in transport or to some other function of GARP. Further investigation of the molecular mechanism of GARP function showed that this complex plays two distinct roles in retrograde transport: (1) tethering of vesicular transport intermediates with the TGN, and (2) interaction with the SNARE proteins, Syntaxin 6, Syntaxin 16 and Vamp4 at the TGN, in a way that promotes their assembly into SNARE complexes. GARP thus orchestrates the tethering and fusion of retrograde transport intermediates by participating in two consecutive, independent steps.